The mitogen-activated protein kinase (MAPK) signaling cascade is activated by oxidants and reactive lipid electrophiles generated by diverse environmental exposures. MAPK signaling is a key driver of environmentally-induced disease processes. The principal upstream trigger for MAPK activation is the MAP kinase kinase kinase (MAP3K) enzyme apoptosis signal-regulating kinase 1 (ASK1), which serves as a key integrating sensor/transducer for MAPK signaling induced by environmental stressors. ASK1 activation requires the assembly of a multiprotein ASK signalosome, which can be regulated in part through ASK1 complexation with reduced thioredoxin 1 (TRX1) and other proteins, including the homologous kinases ASK2 and ASK3. This project addresses the central question of how oxidative stress activates the ASK signalosome. We hypothesize that oxidants and lipid electrophiles oxidize and covalently modify ASK proteins or their interacting partners to destabilize ASK inhibitory complexes and enable assembly of an active ASK signalosome. We will apply quantitative mass spectrometry (MS)-based analyses in human cell models to test this hypothesis through the following specific aims: 1) Define the composition of pre- and post-activation ASK signalosomes. ASK complexes will be analyzed by capture of TAP-tagged ASK1/2/3 and protein partners in 3 human cell models. We will screen a panel of lipid electrophiles and H2O2 for activation of MAPK signaling and then inventory pre- and post-activation ASK signalosomes by shotgun proteomics. We will configure a multiplexed panel of parallel reaction monitoring (PRM) mass spectrometry assays to quantify the components of pre- and post-activation ASK signalosomes. 2) Identify oxidative modifications, electrophile adducts and ubiquitination signatures associated with ASK activation and regulation. We will identify protein components of ASK signalosomes that are targets for electrophile adduction and cysteine thiol oxidation and then map sites of modification by MS/MS. We will develop PRM assays for modified peptides to enable quantitation of phosphorylations, ubiquitination tags, cysteine redox changes, and electrophile adducts. Alkynyl-electrophile probes will allow covalent capture of adducts and cysteine sulfenic acids using Click chemistry methods. 3) Characterize the dynamics of ASK signalosome activation by oxidants and lipid electrophiles. These studies will quantify ASK signalosomes at the level of protein composition, adduction and oxidation as signalosome activation evolves. These studies will establish an all components methodology for multiprotein complex dynamics that will have broad applicability to functional multiprotein systems. 4) Develop prototype reporter systems for monitoring ASK activation in cell models. We will co-express fluorescent reporter-tagged ASK and interacting proteins to establish F?rster resonance energy transfer (FRET) assays for perturbation of key interactions in the ASK signalosome. These models will enable translation of this work into highly specific and molecularly informative standardized screening methods for ASK and MAPK activation by chemicals.